The production of A? peptides occurs throughout life in mammals, and their progressive accumulation in human brain is an invariant and necessary feature in all cases of Alzheimer?s disease. The production of A? requires proteolysis by ?-secretase followed by ?-secretase, and understanding the process underlying these sequential cleavages is fundamental to cell biology. In particular, identifying the mechanism by which holo-APP is cleaved sequentially to A? peptides is critical for designing safe and effective inhibitors and modulators of this process in order to treat and ultimately prevent a major portion of age-related cognitive decline. Using biochemical and cell biological approaches, we recently discovered that APP processing by ?- and ?- secretases can occur in a large, multi-protease fraction that allows for efficient sequential cleavages of substrates within a high molecular weight (HMW) complex stabilized by members of the tetraspanin web(1). This unexpected finding about coordinated ?/? processing raised the question of whether a similar mechanism exists for the ?- and ?-secretase cleavages which generates A? from APP and could create analogous protein fragments from many other ?/? substrates. In the last few years, there has been substantial progress in deciphering the 20-TMD structure of the PS/?-secretase complex(2). However, we still know very little about the cell biological mechanism of the two-step processing that defines RIP. It has been assumed that the post- sheddase CTFs are trafficked to a membrane site where ?-secretase is active(3), but how such presumptive movement within the membrane occurs so that the CTFs correctly finds and enters the docking and active sites of ?-secretase remains a mystery. It is this obligatory, 2-step feature of RIP that we probe in this R03 application by an early stage investigator. To address these mechanisms and also test the feasibility of targeting the ?/? complex, we propose the following two Specific Aims. First, we will confirm and characterize a novel, catalytically active ?/?-secretase complex we recently discovered and isolated from cultured cells, mouse brain and human brain by using 1) protein-protein-interaction approaches including co-IP, native PAGE, FPLC, PLA, NanoBiT (reversible with kinetics) and BiFC (irreversible), and 2) a novel experimental paradigm the PI invented to perform functional enzymatic characterization of ?/?-secretases through a collection of new homemade A? ELISA assays. Second, we will search for regulatory components associated with this ?/?- secretase complex through 1) protein identification and quantitative proteomics analysis of ?/?-secretases complexes isolated from cultured cells and human brain; 2) genetic manipulation of potential hits from proteomic screening to explore complex assembly and stabilization; and 3) test and design small compounds based on the ?/?-mechanism we?ve discovered. The completion of the proposed study will provide mechanistic insights into the coordinated proteolysis by a single protease complex as a previously unrecognized cell biological event, its involvement in AD pathogenesis, and as a target for more specific ?/?-modulators.